The authors thank the Division of Media Support Services, Mayo Clinic Rochester, MN for video production as well as Georgios Kotsougianis for editing of the video. The excellent artwork was conducted by Jim Postier, Rochester, MN. Additionally, the authors wish to thank the German research foundation (Deutsche Forschungsgemeinschaft) for providing salary support for Dr. Dimitra Kotsougiani (DFG grant: KO 4903/1-1). This work was supported by a generous gift from Tarek E. Obaid. This work was performed in the Microvascular Research Laboratory, Department of Orthopedic Surgery Mayo Clinic Rochester, MN.

The study was approved by the Institutional Animal Care and Use Committee (IACUC) at Mayo Clinic Rochester. Yucatan miniature swine were serving as both donors and recipients during this surgical VCA procedure. Pairing of donor and recipient was based upon DNA sequence swine leukocyte antigen (SLA) haplotyping to ensure a major mismatch in the SLAs 22,23. Animals were age- and weight-matched and of identical blood type. Two surgical teams simultaneously harvested...

<ol>
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The authors declare that they have no competing financial interests.

The transplantation of vascularized allogeneic bone (bone VCA) may represent a future reconstructive option for large segmental osseous defects. However, the need of long-term-immunosuppression (IS) and its significant side effects required for bone VCA survival are difficult to justify in these nonlife-critical-applications6.
Although inbred strains of the laboratory rat have been used extensively in allotransplantation research to test various approaches for avoidance...

Large segmental osseous defects result from trauma, infection or limb-sparing surgery after malignancy. Current reconstructive options such as vascularized autogenous bone grafting, bone transport, prosthetic replacement, and cryopreserved necrotic allografts, used alone or in combination, are associated with significant morbidity and have high rates of complications1,2,3.
The presence of a microvascular network is essential for formation and homeostasis of bone, supporting osteogenic, chondrogenic and mesenchymal stem cells required for bone repair4.
The transplantation of living allogeneic bone, a form of vascularized composite tissue allotransplantation (bone VCA), performed with microsurgical anastomosis of its nutrient pedicle, may represent a future reconstructive alternative. Like cryopreserved allogeneic bone, immediate stability is provided by closely matching bone defect morphology. Like autogenous vascularized graft, it provides the enhanced healing and remodeling of living bone tissue. The obstacle in any allotransplant procedure remains the need of long-term-immunosuppression (IS). The problem is more acute in musculoskeletal tissues, which require drug doses 2-3 times greater than organ transplants5. Concomitant risks including organ toxicity, malignancy, infection or development of graft-versus-host disease are difficult to justify in these nonlife-critical-applications6. However, episodes of acute and chronic rejection remain a major issue with current long-term IS7. Ongoing effort to closely match histocompatibility antigens, induce donor-specific tolerance and/or improve drug immunotherapy have not as yet routinely succeeded in permitting clinical drug-free tissue survival8,9.
We have previously demonstrated the means to maintain bone VCA viability and enhance bone remodeling in small animal models by promotion of a new autogenous circulation within transplanted bone. This is done by the additional use of surgical angiogenesis from implanted autogenous tissue10,11,12. Allogeneic bone segments are transplanted microsurgically with anastomosis of the nutrient bone segment pedicle. In addition host-derived vessels are implanted into the medullary canal of the allogeneic vascularized bone segment. During this 2-week process, patency of the allogeneic nutrient vessel is maintained with drug immunosuppression. After IS-withdrawal, the nutrient pedicle will eventually thrombose13. The new capillary bed, based on the host-derived vessels provides sufficient circulation to maintain tissue viability. Bone healing and remodeling are enhanced since osteogenesis and angiogenesis are coupled10,11,12. No further immunotherapy is required and bone viability is maintained long-term despite an immunologically competent host and absence of donor-specific tolerance.
Translation of this novel method of bone allotransplantation into clinical practice should best be preceded by further study of healing, mechanical properties and immunology in a large animal model. The porcine model is ideal for such VCA research14,15,16. Miniature swine are comparable in size and anatomy to man, allowing skeletal reconstruction using essentially identical surgical implants and techniques. Swine immunology is well defined, including swine leukocyte antigen (SLA) haplotypes and blood types, necessary for transplant surgery. Cell lineage studies are possible with sex-mismatched transplantation, as are detailed analyses of immune responses17,18,19,20,21.
Here, we describe a bone VCA allotransplantation model in the Yucatan miniature swine, suitable for study of segmental bone loss and reconstruction. This model can be used to investigate the interplay of surgical angiogenesis and short-term IS on bone VCA survival and function, including osteocyte lineage, bone blood flow, healing and remodeling capacities, alloresponsiveness and biomechanics as well as to test other innovative immune modulatory strategies.

<table border="0" cellpadding="0" cellspacing="0" width="812">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Xylazine</td>
			<td style="width:311px;">VetTek, Bluesprings, MO</td>
			<td style="width:119px;">N/A</td>
			<td style="width:167px;">2mg/kg</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Telazol</td>
			<td style="width:311px;">Pfizer Inc., NY, NY</td>
			<td align="right" style="width:119px;">2103</td>
			<td>5mg/kg</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Buprenorphine</td>
			<td style="width:311px;">Zoo Pharm, Windsor, CO</td>
			<td style="width:119px;">N/A</td>
			<td>0.18mg/kg</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cefazoline</td>
			<td style="width:311px;">Hospira, Lake Forest, IL</td>
			<td style="width:119px;">RL-4539</td>
			<td>1g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ethilon sutures</td>
			<td style="width:311px;">Ethicon, Sommerville, NJ</td>
			<td style="width:119px;">BV 130-5</td>
			<td>9-0</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Locking plate</td>
			<td>DePuy Synthes Vet, West Chester, PA</td>
			<td style="width:119px;">VP4041.09</td>
			<td>9-hole 3.5mm locking plate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vicryl sutures</td>
			<td style="width:311px;">Ethicon, Sommerville, NJ</td>
			<td style="width:119px;">J808T</td>
			<td>2-0, 3-0</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tegaderm</td>
			<td>3M Health Care, St. Paul, MN&#160;</td>
			<td align="right" style="width:119px;">16006</td>
			<td>15x10cm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hickman catheter</td>
			<td style="width:311px;">Bard Access System Inc., Salt Lake City, UT</td>
			<td align="right">600560</td>
			<td>9.6 French</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Carprofen</td>
			<td style="width:311px;">Zoetis Inc., Kalamazoo, MI</td>
			<td>1760R-60-06-759</td>
			<td>4mg/kg</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tacrolimus</td>
			<td>Sandoz Inc., Princeton, NJ&#160;</td>
			<td align="right" style="width:119px;">973975</td>
			<td>(0.8-1.5mg/kg/day)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Mycophenolate Mofetil&#160;</td>
			<td>Sandoz Inc., Princeton, NJ&#160;</td>
			<td align="right">772212</td>
			<td>(50-70mg/kg/day)&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Methylprednisolone sodium succinate</td>
			<td>Pfizer Inc., NY, NY</td>
			<td style="width:119px;">2375-03-0</td>
			<td>500 mg</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gentamicin</td>
			<td style="width:311px;">Sparhawk Laboratories, Lenexa, KS</td>
			<td style="width:119px;">1405-41-0</td>
			<td>3mg/kg&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dermabond Prineo</td>
			<td style="width:311px;">Ethicon, San Lorenzo, Puerto Rico</td>
			<td style="width:119px;">6510-01-6140050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Isoflurane 99.9% 250 ml</td>
			<td style="width:311px;">Abbott Animal&#160; Health&#160;</td>
			<td style="width:119px;">05260-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Lactated Ringer&#39;s 1L</td>
			<td style="width:311px;">Baxter Corporation</td>
			<td style="width:119px;">JB1064</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Saline 0.9%, 1 L</td>
			<td>Baxter Corporation</td>
			<td align="right">60208</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ceftiofur</td>
			<td style="width:311px;">Pfizer Canada Inc.</td>
			<td align="right" style="width:119px;">11103</td>
			<td>5mg/kg</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microfil</td>
			<td>Flow Tech Inc, Carver, MA</td>
			<td>MV-122</td>
			<td>125 ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Decalcifying Solution</td>
			<td>Thermo Fisher Scientific, Chesire, WA, UK</td>
			<td style="width:119px;">8340-1</td>
			<td></td>
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surgical angiogenesis in porcine tibial allotransplantation: a new large animal bone vascularized composite allotransplantation model

Segmental bone loss resulting from trauma, infection malignancy and congenital anomaly remains a major reconstructive challenge. Current therapeutic options have significant risk of failure and substantial morbidity.
Use of bone vascularized composite allotransplantation (VCA) would offer both a close match of resected bone size and shape and the healing and remodeling potential of living bone. At present, life-long drug immunosuppression (IS) is required. Organ toxicity, opportunistic infection and neoplasm risks are of concern to treat such non-lethal indications.
We have previously demonstrated that bone and joint VCA viability may be maintained in rats and rabbits without the need of long-term-immunosuppression by implantation of recipient derived vessels within the VCA. It generates an autogenous, neoangiogenic circulation with measurable flow and active bone remodeling, requiring only 2 weeks of IS. As small animals differ from man substantially in anatomy, bone physiology and immunology, we have developed a porcine bone VCA model to evaluate this technique before clinical application is undertaken. Miniature swine are currently widely used for allotransplantation research, given their immunologic, anatomic, physiologic and size similarities to man. Here, we describe a new porcine orthotopic tibial bone VCA model to test the role of autogenous surgical angiogenesis to maintain VCA viability.
The model reconstructs segmental tibial bone defects using size- and shape-matched allogeneic tibial bone segments, transplanted across a major swine leukocyte antigen (SLA) mismatch in Yucatan miniature swine. Nutrient vessel repair and implantation of recipient derived autogenous vessels into the medullary canal of allogeneic tibial bone segments is performed in combination with simultaneous short-term IS. This permits a neoangiogenic autogenous circulation to develop from the implanted tissue, maintaining flow through the allogeneic nutrient vessels for a short time. Once established, the new autogenous circulation maintains bone viability following cessation of drug therapy and subsequent nutrient vessel thrombosis.

The described technique was successfully performed in four SLA major mismatched Yucatan miniature swine and segmental tibial defects reconstructed using size-matched tibial VCA. Simultaneous nutrient vessel repair of the bone allotransplant and implantation of an AV bundle from the recipient animal within the allotransplant medullary canal permitted both immediate bone circulation and development of a new autogenous blood supply over time (Figure 1). At 16 we...

Watch this Scientific Journal Video about Surgical Angiogenesis in Porcine Tibial Allotransplantation: A New Large Animal Bone Vascularized Composite Allotransplantation Model at JoVE.com

Surgical Angiogenesis in Porcine Tibial Allotransplantation: A New Large Animal Bone Vascularized Composite Allotransplantation Model

Currently any kind of vascularized composite allotransplantation depends on long-term-immunosuppression, difficult to support for non-life-critical indications. We present a new porcine tibial VCA model that can be used to study bone VCA and demonstrate the use of surgical angiogenesis to maintain bone viability without the need of long-term immune-modulation.

Segmental bone loss resulting from trauma, infection malignancy and congenital anomaly remains a major reconstructive challenge. Current therapeutic ...

The overall goal of this project is to establish a new large animal model for study of segmental bone loss and reconstruction using vascularized composite tibial allotransplantation. Annually, approximately 3, 000 limb-sparing surgeries are performed in the United States to reconstruct segmental bone defects resulting from neoplasm, infection, congenital anomaly, or trauma. Current reconstructive options are associated with significant mobility and high complication rates.The use of bone allotransplantation may allow replacement of resected bone with size and shape-matched living bone. Allotransplantation of bone has seldom been performed clinically, in part due to the need for long-term immunosuppression to prevent rejection and result in tissue death. Small animal models demonstrate an alternative to drug immunosuppression or tolerance induction.Like any organ or limb allotransplant, blood flow is maintained by microsurgical repair of nutrient vessels. At surgery, an additional unique step is performed, placing a recipient-derived vascular supply within the medullary canal of the transplanted segment. The resulting neoangiogenic blood supply develops rapidly, along cessation of immunosuppressive therapy after two weeks.Prior to clinical use, it is prudent to further study bone allotransplantation in a large animal model as described in this video. In a two-team approach, a pair of two major SLA mismatched Yucatan mini pigs are being operated simultaneously, each pig serving as recipient and donor. First, a tibial bone segment where a vascular pedicle is harvested from the right tibia of each pig.In this process, a segmental tibial defect is created in each pig. In a cross-transplantation manner, the tibial bone segments are exchanged between the two Yucatan mini pigs to reconstruct each tibial bone defect. After placement of the allogenic tibial bone segment and the created tibial bone defect, a recipient-derived arterial venous bundle from the lower hind limb is implanted into the allogenic tibial bone segment to promote a recipient-derived neoangiogenic circulation within the bone segment.To accomplish the vascularization of the allotransplant, a microsurgical anastomosis of the nutrient pedicle of the allogenic tibial bone segment to a muscle branch of the recipient lower hind limb is performed. Bone fixation is achieved with a locking compression plate. In the following two weeks, a short-term immunosuppression is administered to maintain patency of the allogenic nutrient pedicle until a new recipient-derived circulation starting from the implanted arterial venous bundle has been established.Fast Yucatan mini pigs a day prior to the procedure and weigh them for control drug administration. After sedation, place a peripheral catheter for intravenous drug and fluid infusion in an ear vein and administer analgesics and prophylactic antibiotics. Next, place the mini pig in a supine position on a warming pad.Shave the right hind limb and wash the leg three times. Prep and drape in a sterile manner. Use an antimicrobial incision drape to seal all wounds.Monitor blood pressure, pulse rate, body temperature, and respiration rate. Draw an incision line with a marker and make an anterior lateral incision beginning above the knee joint along the anterior ridge of the tibia to the tibiotalar joint. Dissect the skin and the subcutaneous tissue and separate the tibia from the anterior compartment musculature.Next, release a part from the tibialis anterior muscle from its origin. Remove a part of the tibial ridge using an oscillating saw to improve the field of view. Identify the cranial tibial artery and vein to be later used as an arterial venous bundle to promote neoangiogenesis.Next, open the interosseous membrane and identify the descending branch of the cranial tibial artery named caudal tibial artery which gives rise to the nutrient pedicle of the tibial diaphysis. Now dissect with great care the cranial and caudal tibial artery and identify the nutrient pedicle of the tibial diaphysis. Next, tack the pedicle with a micro clamp.Do not detach the vascular pedicle. Identify a muscle branch in the anterior muscle compartment to be later used for the anastomosis of the bone vascularized allotransplant. Next, start the harvest of the tibial bone segment including the vascular pedicle.Use a cutting jig to ensure a pre-sized and reproducible bone resection. Fix the cutting jig on the medial surface of the tibia. Guided by the jig, perform parallel bone cuts in a distance of exactly 3.5 centimeters to gain both an allotransplant and a defect of 3.5 centimeters.Then remove the cutting jig. By rotating the tibial bone segment, visualize the nutrient pedicle of the tibial bone segment. Detach the nutrient pedicle and free the tibial piece carefully leaving a thin muscle cuff on its surface completing the elevation of the tibial bone segment including its vascular pedicle.Once removed, the tibial bone segment is now ready for microvascular transfer. Next, ligate the cranial tibial arterial venous bundle distally for transposition into the allogenic tibial bone segment. Now exchange in a cross-transplantation manner the harvested tibial bone segments between the two major mismatched mini pigs.To allow passage of the cranial tibial arterial venous bundle, remove a V-shaped segment from the proximal tibial osteosynthesis side and drill a hole into the distal osteosynthesis side. Next, introduce the recipient animal's AV bundle into the allotransplant for subsequent development of a new angiogenic circulation. Anastomose the nutrient pedicle of the bone allotransplant to the prepared muscle branch of the tibial anterior compartment in an end-to-end manner.Achieve osteosynthesis using a nine-hole 3.5 locking plate. Perform fascial and layered skin closure. Finally apply an occlusive compressive dressing on the wound.For postoperative immunosuppressive drug administration and monitoring, place a venous catheter into the external jugular vein. Through a longitudinal anterior lateral incision in the neck, expose the vein. By tunneling, insert a Hickman catheter from the back.Place the catheter into the jugular vein and secure it with nonabsorbable sutures. Additionally, secure the catheter to the skin and close the neck layers. Allow the pig to recover for 60 minutes and then monitor closely until completely recovered.Then return the mini pig to its own pen and allow to drink water and eat food ad libidum. For postoperative analgesia, use Carprofen. Additionally, administer prophylactic antibiotics daily for two weeks.To maintain blood flow of the bone vascularized allotransplant through the nutrient pedicle during AV bundle angiogenesis, administer a triple therapy consisting of Tacrolimus, Mycophenolate Mofetil, and Prednisolone for 14 days. Adjust daily doses of immunosuppressive drugs according to aimed blood trough levels. Reduce the initial dose of Prednisolone gradually until the maintenance dose is obtained.The described technique was successfully performed in four SLA major mismatched Yucatan mini pigs. All mini pigs survived until the end of followup and were walking without limping. Analysis of blood samples showed that therapeutic trough levels for Tacrolimus and Mycophenolate Mofetil were achieved.This ensured adequate immunosuppression to prevent nutrient vessel damage and thrombosis due to rejection during the first two weeks postoperative. Thereafter, a new recipient-derived blood supply had been established within the allogenic bone allotransplant, obviating the need for the bone allogenic circulation. A 25-point scoring system was used to quantify VCA incorporation.Regular radiologic evaluation demonstrated progressive healing of the bone PCA over the study period. Internal fixation was maintained without loss of reduction or loosening. We have developed a reliable and reproducible large animal model for bone VCA which is feasible to test for surgical angiogenesis combined with short-term immunosuppression.This model will serve as basis for future studies investigating the interplay between angiogenesis, allotransplant survival, and immune response. Furthermore, it can be used to delineate the complex process of bone VCA rejection and other innovative immune modularity strategies.

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7.5K Views.  Mayo Clinic. The overall goal of this project is to establish a new large animal model for study of segmental bone loss and reconstruction using vascularized composite tibial allotransplantation. Annually, approximately 3, 000 limb-sparing surgeries are performed in the United States to reconstruct segmental bone defects resulting from neoplasm, infection, congenital anomaly, or trauma. Current reconstructive options are associated with significant mobility and high complication rates.The use of bo...